ABSTRACT-PROJECT 2 Principal Investigator: Terry R. Brown Numerous studies have demonstrated that the androgen receptor (AR) and its ligand, testosterone (T), are required for normal spermatogenesis. Recently published observations showing that mice with Sertoli cell specific conditional knockout of the AR gene do not produce sperm provide definitive proof that AR expression in Sertoli cells is absolutely required for male fertility. However, despite the importance of the AR in the regulation of spermatogenesis, there has been no comprehensive effort to understand how AR expression itself is regulated. The focus of this application is to define the molecules and mechanisms that drive expression of the AR gene in Sertoli cells. Sertoli cells in rats, mice and men exhibit highly concordant patterns of AR expression. During pubertal maturation, when spermatogenesis becomes an increasingly efficient process, there is an increase in AR expression in Sertoli cells. In adulthood, the pattern of AR expression in Sertoli cells is synchronized with the progression of the neighboring spermatogenic cells through the stages of the cycle of the seminiferous epithelium. Importantly, the stages with the highest level of expression of the AR gene are the most sensitive to the loss of androgen stimulation. Thus, the central hypothesis of this proposal is that the expression of AR by Sertoli cells, both during pubertal maturation and in the adult, is determined by the activity of a small subset of transcription factors and their cognate cis-acting regulatory elements within the AR gene. Important among these molecules are those that regulate the maturational and stage-specific changes in AR gene transcription. The .three specific aims of this proposal will test this hypothesis by identifying the transcription factors and cognate cis-acting regulatory elements in the rat AR gene promoter that are required for maturation-dependent and stage-specific expression of AR by Sertoli cells and determine if those elements and factors are required for the in vivo expression of AR by Sertoli cells. Population control, as well as reproductive success and failure, remain significant issues for public health. Thus, our proposed experiments should reveal important mechanisms that regulate AR expression not only in rodents but also in humans, and thereby contribute to our understanding of male fertility.